through marker-assisted selection of ahfad2 alleles in
TRANSCRIPT
Improving oil quality by altering levels of fatty acidsthrough marker-assisted selection of ahfad2 alleles in peanut(Arachis hypogaea L.)
Sandip K. Bera . Jignesh H. Kamdar . Swati V. Kasundra .
Pitabas Dash . Anil K. Maurya . Mital D. Jasani . Ajay B. Chandrashekar .
N. Manivannan . R. P. Vasanthi . K. L. Dobariya . Manish K. Pandey .
Pasupuleti Janila . T. Radhakrishnan . Rajeev K. Varshney
Received: 4 May 2018 / Accepted: 11 August 2018
� Springer Nature B.V. 2018
Abstract Peanut plays a key role to the livelihood of
millions in the world especially in Arid and Semi-Arid
regions. Peanut with high oleic acid content aids to
increase shelf-life of peanut oil as well as food
products and extends major health benefits to the
consumers. In peanut, ahFAD2 gene controls quantity
of two major fatty acids viz, oleic and linoleic acids.
These two fatty acids together with palmitic acid
constitute 90% fat composition in peanut and regulate
the quality of peanut oil. Here, two ahfad2 alleles from
SunOleic 95R were introgressed into ICGV 05141
using marker-assisted selection. Marker-assisted
breeding effectively increased oleic acid and oleic to
linoleic acid ratio in recombinant lines up to 44% and
30%, respectively as compared to ICGV 05141. In
addition to improved oil quality, the recombinant lines
also had superiority in pod yield together with desired
pod/seed attributes. Realizing the health benefits and
ever increasing demand in domestic and international
market, the high oleic peanut recombinant lines will
certainly boost the economical benefits to the Indian
farmers in addition to ensuring availability of high
oleic peanuts to the traders and industry.
Keywords Peanut �Oleic acid �Oil quality �Marker-
assisted selection (MAS) � ahFAD2 gene
Electronic supplementary material The online version ofthis article (https://doi.org/10.1007/s10681-018-2241-0) con-tains supplementary material, which is available to authorizedusers.
S. K. Bera (&) � J. H. Kamdar � S. V. Kasundra �P. Dash � A. K. Maurya � M. D. Jasani �A. B. Chandrashekar � T. RadhakrishnanIndian Council of Agricultural Research-Directorate of
Groundnut Research (ICAR-DGR), Junagadh, India
e-mail: [email protected]
N. Manivannan
National Pulses Research Center, Tamil Nadu
Agricultural University (TNAU), Pudukkottai, India
R. P. Vasanthi
Regional Agricultural Research Station, Acharya NG
Ranga Agricultural University (ANGRAU), Tirupati,
India
K. L. Dobariya
Main Oilseeds Research Station, Junagadh Agricultural
University (JAU), Junagadh, India
M. K. Pandey � P. Janila � R. K. VarshneyInternational Crops Research Institute for the Semi-Arid
Tropics (ICRISAT), Hyderabad, India
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Euphytica (2018) 214:162
https://doi.org/10.1007/s10681-018-2241-0(0123456789().,-volV)(0123456789().,-volV)
Introduction
Peanut (Arachis hypogaea L.) is cultivated in an area
of * 25.44 m ha with a production of * 45.22 m
tons (FAO 2014). Based on average production, the
China, India, Nigeria, United States of America and
Sudan are top five peanut producing countries in the
world which together contribute * 70% to the global
peanut production (FAO 2014). In USA and other
European countries, major share of peanut produce
(* 75%) goes towards confectionary and other food
purpose, while half of the produce (* 49%) goes for
oil extraction in two major groundnut producing
countries viz, China and India. Fatty acid composition
defines the peanut oil quality which contains 80%
unsaturated fatty acids (UFA) and 20% saturated fatty
acids (SFA). Oleic and linoleic acids constitute UFA,
while palmitic, stearic, arachidic, gadoleic, behenic
and lignoceric acids constitute SFA. Palmitic acid
alone constitutes about 10% of SFA, while remaining
five fatty acids together constitute remaining 10% of
SFA (Kavera et al. 2014). Together oleic, linoleic and
palmitic acids comprise * 90% of the total fatty acid
composition and regulate the quality of peanut oil
(Moore and Knauft 1989).
Peanut oil is preferred for cooking purpose due to
its higher UFAs to SFAs ratio (Johnson and Saikia
2008). In general SFAs increase serum low-density
lipoproteins (LDL) cholesterol level in the blood.
Excess consumption of palmitic acid increases the risk
of cardiovascular diseases (CVD) (WHO 2003). On
the other hand higher linoleic acid in oil helps in
oxidative rancidity and oil becomes thermodynami-
cally unstable upon heating (Kratz et al. 2002).
Furthermore, variation in linoleic acid content pro-
motes formation of trans-fatty acid which also causes
CVD. In contrast, high oleic acid content in cooking
oil decreases the risk of CVD by reducing the level of
serum LDL cholesterol and maintains level of high-
density lipoproteins (HDL) (Rizzo et al. 1986; Wang
2009; Vassiliou et al. 2009). Besides, consumption of
peanut products rich in oleic acid decreases tumori-
genesis, and ameliorate inflammatory diseases
(O’Byrne et al. 1997; Yamaki et al. 2005). Oleic acid
has 10-fold higher auto-oxidative stability than
linoleic acid (O’Keefe et al. 1993). Thus, peanut and
its byproducts with high oleic acid as well as high oleic
to linoleic acid ratio (O/L ratio) have longer shelf life
than normal peanut (Mozingo et al. 2004). Therefore,
breeding peanut varieties with high oleic acid and
reduced level of linoleic and palmitic acids are
essential to make peanut nutritionally more desirable
to the consumers (Janila et al. 2016). Availability of
F435, a peanut mutant with 80% oleic acid and 2%
linoleic acid contents, helped in breeding high oleic
peanuts, SunOleic 95R followed by SunOleic 97R in
USA (Norden et al. 1987). Previous studies have
reported that two alleles each in the A-genome
(ahFAD2A) and in the B-genome (ahFAD2B) control
fatty acids contents in peanut (Wang et al.
2013, 2015a). Subsequently, Chu et al. (2009) and
Chen et al. (2010) developed linked cleaved amplified
polymorphic sequence (CAPS) and allele-specific
markers, respectively for both ahFAD2A and
ahFAD2B alleles. Associated CAPS markers to high
oleic acid in peanut helped to breed ‘Tifguard High
O/L’ variety in USA (Chu et al. 2007). Recently,
Janila et al. (2016) developed high oleic peanuts using
both AS-PCR and CAPS markers. Here we used both
AS-PCR and CAPS markers for breeding high oleic
recombinants of ICGV 05141. The female parent,
ICGV 05141 used here is different from Janila et al.
(2016) and has high pod and oil yield together with
desirable pod and seed features. High oleic peanut
recombinant lines bred here are of different genetic
background and under large-scale yield trials which
would be released soon for cultivation to the Indian
peanut farmers.
Materials and methods
Plant material
The ICGV 05141, a Virginia bunch genotype (A. hy-
pogaea ssp. hypogaea var. hypogaea), is derived from
the cross {{[(Robut 33-1 9 NC Ac 316) 9 (Robut
33-1 9 CS 9)] 9 ICGV 93023} 9 ICGV 99160}.
ICGV 05141, a high oil (55.1%) and normal oleic
acid content (55.8%) peanut breeding line was used as
female parent. ICGV 05141 is one of the 52 high oil
containing peanut breeding line developed by ICRI-
SAT and tested for yield at ICAR–Directorate of
Groundnut Research (ICAR-DGR), Junagadh, Gujarat
over four seasons. Pod yield of ICGV 05141 was
2672 kg/ha and 2416 kg/ha during 2011 rainy and
2012 rainy seasons, respectively. While, in 2011 post
rainy and 2012 post rainy seasons pod yield was
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162 Page 2 of 15 Euphytica (2018) 214:162
1486 kg/ha and 1875 kg/ha, respectively (Project
report, ‘Development and promotion of….Groundnut
farmers in India’ ICRISAT, 2012; unpublished). The
SunOleic 95R was used as male parent which contains
ahfad2a and ahfad2b alleles and characterized with
low oil (45%) and high oleic acid (* 80%) containing
line. The SunOleic 95R was bred using F435 mutant at
Florida Experimental Agriculture Station, USA and
was characterized as a Virginia runner peanut (Gorbet
and Knauft 1997). However, in our experiment we
found Virginia bunch growth habit in SunOleic 95R.
Molecular markers
Breeding population in early generations (F1 and F2)
was screened with two types of markers linked to
ahFAD2 gene. Plants with wild or mutant alleles were
identified using the allele specific-polymerase chain
reaction (AS-PCR) markers (Chen et al. 2010). While,
plants with homozygous or heterozygous alleles were
identified using the cleaved amplified polymorphic
sequences (CAPS) markers (Chu et al. 2009) (Supple-
mentary Table).
DNA extraction and marker genotyping
Tender leaf samples from 10 to 15 days old seedlings
were collected. The DNA was extracted from the leaf
samples of ICGV 05141, SunOleic 95R and segregat-
ing progenies using modified cetyltrimethyl ammo-
nium bromide (CTAB) extraction method (Mace et al.
2003). The quality of DNA was tested on 0.8%
agarose gel (Lonza, USA). The concentration of DNA
was checked in ND100 Spectrophotometer (Nano
Drop Technology, USA) and later concentration was
normalized to * 100 ng/ll for downstream
application.
Genotyping with allele specific-polymerase chain
reaction markers
Amplification of ahfad2a and ahfad2b alleles were
checked using two different primer pairs, while a
separate primer pair was used to amplify ahFAD2A or
ahFAD2B alleles (Chen et al. 2010). In case of mutant
allele (substitution from G:C?A:T) in the A-genome
the primer combination, F435-F and F435SUB-R
amplified a 203 bp fragment, while in case of mutant
allele (A:T insertion) in the B-genome the primer
combination, F435-F and F435INS-R amplified a
195 bp fragment. For wild type allele, the primer
combination, F435-F and F435WT-R amplified a 193
bp fragment. F435-F and F435IC-R, amplified a
250 bp fragment and used as internal control to
confirm successful amplification of alleles. The poly-
merase chain reaction (PCR) for AS-PCRmarkers was
carried out in C1000 Thermal cycler (BIO-RAD,
USA). The PCR reaction was setup in 25 ll volume
using the protocol of KAPA3G Plant PCR Kit
(KK7251, Kapa Biosystems, USA). Amplification of
PCR assay was done using protocol as mentioned in
Janila et al. (2016). The amplified DNA fragments
along with 100 bp DNA marker (Thermo Scientific,
USA) were size separated on a 2.0% horizontal
agarose gel (Lonza, USA). Gel electrophoresis was
carried out in 1X TBE buffer at 100 V current for one
to two hours. The Ethidium bromide was used for
staining the DNA fragments and gel was scanned
using laser scanner (Fujifilm FLA 5100, Japan) for
scoring.
Genotyping with CAPS markers
The PCR amplification was carried out in BIO-RAD
C1000 Thermal cycler. For amplification of
ahFAD2A/ahfad2a alleles the primer pairs aF19F
and 1056R (IDT, USA) were used. Amplified product
was further digested with Hpy99I (New England Bio
Labs, UK) restriction enzyme having single recogni-
tion site to detect 448 G?A mutation. Similarly, the
primers bF19F and R1/FADR (IDT, USA) were used
to detect insertion mutation (insertion of single base
‘A’) at 441_442 bp in the ahFAD2B/ahfad2b alleles.
The PCR reaction was setup in 25 ll volume using the
protocol of KAPA3G Plant PCR Kit (KK7251, Kapa
Biosystems, USA). Amplification of PCR assay was
done using protocol as mentioned in Janila et al.
(2016) and Chu et al. (2009). The PCR product was
resolved on 2% agarose gel for confirming the
amplification and digested with restriction enzyme
after purification. Restriction digestion of the 10 ll ofA-genome amplicon was done using 0.5 U of restric-
tion enzyme Hpy99I (New England Biolabs, UK) by
incubating at 37 �C for about 4 h. In case of ahFAD2A
allele, the 826 bp fragment was digested to 598 bp and
228 bp, while ahfad2a allele had the 826 bp fragment
intact. On the other hand, the restriction digestion of
10 ll of B-genome amplicon was done using 2.0 U of
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Euphytica (2018) 214:162 Page 3 of 15 162
restriction enzyme Hpy188I (New England Biolabs,
UK) by incubating about 16 h at 37 �C. The 1214 bp
ahFAD2B allele having five restriction sites cleaved
into five fragments i.e., 736, 263, 171, 32 and
12 bp. While in case of ahfad2b allele, the 736 bp
fragment had one additional restriction site which
further cleaved into 550 and 213 bp. Thus, ahfad2b
allele produced six fragments instead of five in case
of ahFAD2B allele.
Back ground testing of recombinant lines was done
with nine simple sequence repeat markers (SSRs).
Nine SSRs (Supplementary Data) were picked up
within the 20 cM of ahFAD2 loci (Gautami et al.
2012). SSRs analysis was done in female parent and 11
recombinant lines (HOP-IL_MAS-108, HOP-IL_MAS-
111, HOP-IL_MAS119, HOP-IL_MAS-120, HOP-
IL_MAS-130, HOP-IL_MAS-154, HOP-IL_MAS-181,
HOP-IL_MAS-201, HOP-IL_MAS-123, HOP-IL_MAS-
144, and HOP-IL_MAS-171). Recombinant lines used
in background testing were selected based on existing
DNA samples in the lab. Amplification of PCR assay
was done using protocol as mentioned in Bera et al.
(2016). The PCR product was resolved on 2% agarose
gel and was scanned using laser scanner (Fujifilm FLA
5100, Japan).
Hybridization and development of MAS lines
Hybridization was done at ICRISAT, Patancharu,
Telangana, India during 2011 rainy season. The F1s
were planted in pots kept inside net-house at ICAR-
DGR, Junagadh, India during 2011 post rainy season.
Individual F1 plants were tagged and genotyped with
allele specific markers. The F1 plants having double
mutant alleles were selfed and harvested single plant
basis. F2 onwards plants were planted in open field of
ICAR-DGR.
A total of 204 F2 plants were planted in 2012 rainy
season and were genotyped with allele specific
markers to identify double mutant lines. Further
double mutant lines were genotyped using CAPS
markers to select homozygous double mutant lines.
Homozygous double mutant lines were further
advanced to next generations during 2012 post rainy
season. A total of 21 homozygous double mutant
single plant progenies were advanced to F4 generation
in 2013 rainy season and subsequently phenotyped for
oil content, protein content and fatty acid profile.
Phenotyping for oil, protein and fatty acid profile was
repeated for entire 21 lines in F5 generation in 2013
post rainy season.
Field evaluation
Selected high oleic recombinant lines were compared
with female parent and elite cultivar for yield and its
related traits in 2014 rainy (F6) and 2015 rainy (F7)
seasons. Recombinant lines, 21 in numbers, together
with female parent (ICGV 05141) and an elite cultivar
(GG 20) were planted in the farm of ICAR-DGR,
Junagadh. Sowing of experiment was done in ran-
domized block design (RBD) with three replications in
second week of June 2014 and harvested in last week
of October 2014. Each genotype was planted in four
lines on four-metre beds. Line to line and plant to plant
spacing were 45 and 10 cm, respectively. Recom-
mended local crop management practices were fol-
lowed for raising a healthy crop. Pod yield per plot
(plot size was 7.2 m2) was recorded at harvest. The
experiment was repeated in 2015 rainy season follow-
ing experimental design and crop management prac-
tices of previous season.
Characterization of genotype
Recombinant lines and ICGV 05141 were character-
ized based on 16 qualitative, 17 quantitative and two
special features following peanut-descriptor (Interna-
tional Board for Plant Genetic Resources (IBPGR) and
International Crops Research Institute for the Semi-
Arid Tropics (ICRISAT) 1992) from five plant
samples collected from field at vegetative, reproduc-
tive and harvesting stages.
Biochemical analysis for oil content and fatty acids
Sound matured kernels (10–15 g) harvested from F3–4and F4–5 progenies were used for both oil content and
fatty acid analysis. The fatty acid composition and oil
content were estimated using Gas chromatography
(GC 700, Thermo Fisher, USA) with flame ionization
detector (FID). For estimation of the esters of fatty
acids the fatty acid methyl esters were passed through
capillary column (TR-wax) (Misra and Mathur 1998).
The FID detector was set to 240 �C and oven at
190 �C. Carrier gas (nitrogen) and fuel gas (hydrogen)were maintained at 30 ml per min. Each sample was
run for 12 min and the peaks were identified by
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162 Page 4 of 15 Euphytica (2018) 214:162
comparison to a FAME standard mix RM-3 (Sigma-
Aldrich, St. Louis, MO).
Statistical analysis
Mean differences among genotypes were done using
Cropstat version 7.2 (IRRI 2007).
Results
Development of MAS progenies
Cross-seed, 11 in numbers, from a cross between
ICGV 05141 and SunOleic 95R were collected from
ICRISAT, Patancheru and were planted at ICAR-
DGR, Junagadh. Upon genotyping of F1s, eight plants
with ahfad2a and ahfad2b alleles were identified. Here
after, plants carrying both ahfad2a and ahfad2b alleles
shall be referred as double mutant plant(s). All eight
double mutant plants were selfed and advanced to F2generation.
A total of 204 F2 plants were grown and genotyped
with allele-specific and CAPS markers (Figs. 1, 2). Of
these, 21 plants were homozygous double mutant
plants and were selfed from F3 generation to F5generation. Upon phenotyping for oil and oleic acid
content consecutively for two years 21 recombinant
lines with high oil and oleic acid content were
selected. Selected recombinant lines and female
parent were further screened with nine SSRs for
selecting ideal recombinants. Out of which PM-170,
AC3C07, TC5D06, Seq4G02, Seq7G02, GM2120,
GM1893 markers amplified both in female parent and
majority of the recombinant lines revealing transfer of
the genomic region adjacent to ahFAD2 loci from
ICGV 05141 to the recombinant lines (Fig. 3).
Oil protein and fatty acid analysis
of recombinant lines and their parents
A significant variation in oil content was observed
among the recombinant lines and their parents which
ranged from 49.7 to 57.9% with an average of 53.3%
during 2013 rainy season (Table 1). Oil content in
majority of the recombinant lines was at par with the
female parent except HOP-IL_MAS-116, HOP-
IL_MAS-120 and HOP-IL_MAS-172 which had 49.7,
50.5 and 50.9% of oil, respectively. During 2013 post
rainy season, oil content ranged from 45.0 to 57.6%
with an average of 53.0% (Table 1). Unlike 2013 rainy
season, majority of recombinant lines were at par with
the female parent except HOP-IL_MAS-116, HOP-
IL_MAS-119, HOP-IL_MAS-172 and HOP-IL_MAS-201
which had 49.3, 46.2, 45.0 and 46.3% of oil, respec-
tively. All the recombinant lines had oil content at par
with female parent except HOP-IL_MAS-116 and
HOP-IL_MAS-172 which had 49.5% and 48.0% of
oil, respectively. Pooled protein content in ICGV
05141 and SunOleic 95R were 25% and 26%,
respectively. Nevertheless, a significant variation
was observed for protein content among recombinant
lines which varied from 21.25 to 25.01% (Table 1).
ICGV 05141 had 55.7% oleic acid, 23% linoleic acid
and 10.8% palmitic acid, while SunOleic 95R had
78.8% oleic acid, 3.7% linoleic acid and 7.6% palmitic
acid. Oleic acid content in recombinant lines ranged
from 57.8 to 80.5% during 2013 rainy season. While,
in 2013 post rainy season, it varied from 55.6 to 82.0%
(Figs. 4, 5). Stable quantity of oleic acid content in
Fig. 1 AS-PCR assay; a Showing amplification of ahfad2a
allele specific 203 bp fragmentin 1 to 4 F1 plants b Amplifica-
tion of ahfad2b allele specific 195 bp fragment in 3 and 4,
while absent in 1 and 2 F1 plants; Where SUN: SunOleic 95R,
M:100 bp DNA ladder
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Euphytica (2018) 214:162 Page 5 of 15 162
both the seasons was noticed in majority of the
recombinant lines, while it varied marginally in a few
of them including SunOleic 95R over seasons. Based
on oleic acid content pooled over two seasons,
majority of the recombinant lines had[ 70% oleic
acid except HOP-IL_MAS-123, HOP-IL_MAS-144,
HOP-IL_MAS-164, HOP-Il_MAS-166 and HOP-
IL_MAS-171 which had 61.0, 58.3, 56.7, 63.8 and
69.1% of oleic acid, respectively (Fig. 6). Wide range
of variation for linoleic acid content was observed
among recombinant lines during both 2013 rainy
season and 2013 post rainy season. It varied from 2.3
to 21.6% in rainy season while from 2.2 to 24% in post
rainy season. Indeed, majority of recombinant lines
had\ 10% linoleic acid (average of two seasons)
except HOP-IL_MAS-123, HOP-IL_MAS-144, HOP-
IL_MAS-164, HOP-IL_MAS-166 and HOP-IL_MAS-171
which had 19.4%, 21.45%, 22.8% 16.9% and 12.25%
of linoleic acid, respectively. Similar to oleic acid,
linoleic acid content also differed between seasons in a
few recombinant lines. While, linoleic acid content in
ICGV 05141 and GG 20 remained almost stable be-
tween seasons. Besides, lower and comparatively
stable linoleic acid content was observed in 10
recombinant lines irrespective of seasons (Table 2).
In case of palmitic acid, marginal variation was
observed in recombinant lines, irrespective of seasons.
Over all, palmitic acid, pooled over two seasons,
varied from 6.6 to 10.5% among recombinant lines
(Fig. 6). We observed up to 44.2% increase in oleic
Fig. 2 CAPS assay;
a Showing heterozygous
and homozygous plants for
ahfad2a allele. b Showing
heterozygous and
homozygous plants for
ahfad2b mutant allele.
Where SUN: SunOleic 95R,
M: 100 bp DNA ladder, CO:
Control, ‘AA, BB’:
homozygous wild alleles,
‘Aa, Bb’: heterozygous
alleles and ‘aa, bb’:
indicates homozygous
mutant alleles
Fig. 3 SSR assisted background selection, Where A—gi-1107,
B—PM-170, C—AC3C07, D—TC5D06, E—Seq 4G02, F—
Seq 7G02, G—GM2120, H—GM1893, I—Seq 17C09. P—
ICGV 05141, M—100 bp ladder, 1 to 11—Recombinant lines
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162 Page 6 of 15 Euphytica (2018) 214:162
acid, while up to 89% and 39.06% decrease in linoleic
and palmitic acid, respectively, in recombinant lines,
as compared to ICGV 05141 (Table 2). Furthermore,
these three fatty acids constituted * 90% of total fat
composition, and palmitic acid content remained
almost constant (in between 6% and 10%) in recom-
binant lines and parents. Indeed peanut genotypes with
both high oleic acid and high oleic to linoleic acid (O/
L) ratio are more desirable. We observed O/L ratio of
15.8 in SunOleic 95R and 2.4 in ICGV 05141, while it
varied from 2.5 to 30.9 in recombinant lines. Out of
which nine lines (HOP-IL_MAS-111, HOP-IL_MAS-
116, HOP-IL_MAS-119, HOP-IL_MAS-130, HOP-
IL_MAS-138, HOP-IL_MAS-145, HOP-IL_MAS-172,
HOP-IL_MAS-191, HOP-IL_MAS-201) had O/L ratio
more than 15.8 along with * 80% oleic acid content
(Fig. 7).
Pod yield
Pooled pod yield/plot was 1453 kg and 1323 kg in
ICGV 05141 and GG 20, respectively, while it varied
from 722 to 2151 kg among recombinant lines. Wide
variation in pod yield was observed between geno-
types as well as seasons. Significant yield superiority
over female parent was observed in five recombinant
lines (HOP-IL_MAS-130, HOP-IL_MAS-145, HOP-
IL_MAS-163, HOP-IL_MAS-181 and HOP-IL_MAS-
Table 1 Oil and protein content estimated at 5% of moisture in recombinant lines and parents
Genotype Oil (%) Protein (%)
2013 rainy 2013 post rainy Pooled 2013 rainy 2013 post rainy Pooled
HOP-IL_MAS-108 53.3 56.8 55.1 24.3 23.1 23.7
HOP-IL_MAS-109 57.2 55 56.1 24.1 23.8 24.0
HOP-IL_MAS-111 52.4 56 54.2 23.8 22.4 23.1
HOP-IL_MAS-116 49.7 49.3 49.5 22.4 23.1 22.8
HOP-IL_MAS-119 56.3 46.2 51.2 23.6 21.0 22.3
HOP-IL_MAS-120 50.5 55.6 53.1 21.8 21.7 21.8
HOP-IL_MAS-123 52.4 51.3 51.9 25.6 24.4 25.0
HOP-IL_MAS-125 53.7 55.6 54.7 23.4 23.4 23.4
HOP-IL_MAS-130 54.8 54.6 54.7 21.7 22.1 21.9
HOP-IL_MAS-138 52.5 55.5 54 23.9 23.3 23.6
HOP-IL_MAS-144 53.7 54.1 53.9 23.3 21.2 22.2
HOP-IL_MAS-145 51.8 57.2 54.5 21.5 21.2 21.4
HOP-IL_MAS-154 52.6 54.5 53.5 22.9 23.1 23.0
HOP-IL_MAS-163 52.7 52.5 52.6 23.6 22.5 23.1
HOP-IL_MAS-164 51.5 55.7 53.6 24.0 24.6 24.3
HOP-IL_MAS-166 57.9 57.6 57.7 22.8 23.0 22.9
HOP-IL_MAS-171 53.3 52.5 52.9 23.0 23.6 23.3
HOP-IL_MAS-172 50.9 45 48 22.1 22.6 22.3
HOP-IL_MAS-181 51.6 50.4 51 22.6 22.7 22.6
HOP-IL_MAS-191 53.2 53.1 53.2 21.3 21.2 21.2
HOP-IL_MAS-201 57.2 46.3 51.8 22.4 20.8 21.6
ICGV 05141 55.1 54.3 54.7 24.6 25.5 25.0
SunOleic 95R 50.9 50.4 50.6 25.1 27.2 26.2
Mean 53.3 53.0 53.2 23.2 22.9 23.1
SE mean 1.42 1.56 1.19 1.27 1.18 1.14
CD (0.05%) 4.04 4.45 3.38 3.62 3.38 3.25
CV% 4.61 5.1 3.87 9.51 8.94 8.56
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191) (Table 3). Besides, additional five recombinant
lines (HOP-IL_MAS-116, HOP-IL_MAS-144, HOP-
IL_MAS-171, HOP-IL_MAS-172 and HOP-IL_MAS-
201) yielded at par with the female parent. Indeed,
these 10 recombinant lines had yield superiority over
GG 20. There was no significant difference in shelling
percent between ICGV 05141 (68%) and GG 20
(66%), while it varied from 65 to 74% among
recombinant lines.
Recombinant lines having superior or at par pod
yield with ICGV 05141 also had superior or at par
shelling percent with female parent except HOP-
IL_MAS-181 which had 65% shelling percent. Simi-
larly, no significant variation in hundred kernel weight
was observed between ICGV 05141 (38 g) and GG 20
(39 g), while it varied from 26 to 39 g among
recombinant lines (Table 3).
Passport data of high oleate genotype
ICGV 05141 is a Virginia bunch genotype with
decumbent-3 growth habit; alternate branching; dark
green colour ovate shape leaf and simple inflores-
cence. The genotype produces 50% flowering at
26 days after germination and matures in 120 days.
Average plant height is 36.0 cm; produces average six
primary branches/plant and two to three flowers/
inflorescence. Average leaflet length and width are
0.010.020.030.040.050.060.070.080.090.0
100.0
Oleic % Linoleic % Palmi�c %
Fig. 4 Oleic, linoleic and
palmitic acid content in
recombinant lines and
parents during 2013 rainy
season
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Oleic % Linoleic % Palmi�c %
Fig. 5 Oleic, linoleic and
palmitic acid content in
recombinant lines and
parents during 2013 post
rainy season
123
162 Page 8 of 15 Euphytica (2018) 214:162
36.6 mm and 14.6 mm, respectively. Pods are mostly
two seeded and average length and width of pods are
36.6 mm and 26.0 mm, respectively. Kernels are rose
in colour, and average length and width of kernels are
28.0 mm and 16.0 mm, respectively (Fig. 8). It yields
202.0 g of pods per square meter area with 32%
harvest index, 70% shelling, hundred kernels weight
of 38 g, * 55% oil content and * 55.7% oleic acid
content. All the recombinant lines developed under
this study were Virginia bunch in growth habit. For
qualitative traits, not much variation was observed
between recombinant line and female parent. How-
ever, moderate to wide variation was observed
between recombinant line and female parent and
between recombinant lines in terms of quantitative
traits and special features. Recombinant lines mostly
differed in days to maturity, plant height, leaflet size,
pod size and kernel size; besides pod yield, shelling
percent and 100-kernel weight. In case of two special
features, majority of recombinant lines were in
combination of high oil content similar to ICGV
05141 and high oleic acid content similar to SunOleic
95R which revealed the success of marker assisted
selection (Supplementary Data).
Discussion
In general, high oleic peanuts are preferred by the
stakeholders due to its enhanced shelf life and multiple
health benefits. Hence, improvement of oleic acid
content in peanut is one of the important breeding
objective worldwide (Janila et al. 2016). The fatty acid
desaturase enzyme catalyzes the conversion of
oleic acid to linoleic acid, and is encrypted by
ahFAD2A and ahFAD2B homeologous alleles (Jung
et al. 2000a, b; Yu et al. 2008). Both the ahFAD2
alleles have 99% sequence homology (Jung et al.
2000a; Lopez et al. 2000) and inactivation of both the
alleles stops conversion of oleic acid to linoleic acid,
causing increased accumulation of oleic acid in peanut
(Jung et al. 2000a, b). Development of molecular
markers linked to ahFAD2 genes has expedited the
breeding high oleic acid content peanut more precisely
in much shorter time and resources. Moreover, MABC
breeding ensures transfer of desired gene/QTL keep-
ing the other features of the female parent intact
(Pandey et al. 2012; Varshney et al. 2013). Previously,
nematode resistance (Simpson et al. 2003a), high oleic
acid content (Chu et al. 2011; Janila et al. 2016) and
rust resistance (Varshney et al. 2014) were transferred
to elite peanut cultivars through molecular breeding.
Transfer of high oleate trait into popular peanut
genotypes has been achieved using both conventional
and molecular breeding. The F435, a mutant with 80%
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Oleic % Linoleic % Palmi�c %
Fig. 6 Oleic, linoleic and
palmitic acid content in
recombinant lines and
parents pooled over 2013
rainy and 2013 post rainy
seasons
123
Euphytica (2018) 214:162 Page 9 of 15 162
oleic acid content was the primary source for high
oleate trait (Norden et al. 1987) and subsequently high
oleate peanut lines such as, SunOleic 95R (Gorbet and
Knauft 1997) and Tamrun OL01 (Simpson et al.
2003b) were developed using F435 through conven-
tional breeding methods. Chu et al. (2007, 2009) first
developed the molecular markers for ahFAD2 genes
and used them for increasing the high oleate trait in
peanut using molecular breeding (Chu et al. 2011;
Janila et al. 2016). Both CAPS and SNP markers,
linked to high oleic acid content, were used to transfer
the mutant alleles. Further selected lines were con-
firmed by HybProbeSNP assay (Bernard et al. 1998).
In a separate study, Mienie and Pretorius (2013)
selected heterozygous and homozygous lines for both
the mutant alleles using multiplex real-time PCR
assay, developed by Barkley et al. (2010). Thus, above
studies showed that linked marker could be conve-
niently used to identify plants, carrying mutant alleles,
in early generations of breeding program aimed at
increasing oleic acid content. Furthermore, the time
and the volume of breeding material in segregating
generations reduced considerably. In our study ICGV
05141, a high oil containing peanut genotype, was
targeted for improving its oil quality using marker
assisted selection. Selection of breeding lines with
desirable oil content and high oleic acid content was
carried out by genotyping in early generations and
phenotyping in advanced generations. Genotyping-
based selection was done in early generations, to
confirm plants with desirable genomic region. Subse-
quently, desirable lines were phenotyped in F4 and F5generations for confirmation. High oil content and
high oleic acid content peanuts are most suitable for
oil industry to produce higher oil production along
with improved oil quality. Furthermore, increased
shelf-life and health benefits of food products made
from high oleic peanuts are boon to all the stake
holders including consumers. In this study we devel-
oped high oleic acid and oil content recombinants
Table 2 Change in oleic acid, linoleic acid and palmitic acid content in recombinant lines with respect to ICGV 05141
Recombinant line Oleic
acid %
% increase in
oleic acid
Linoleic acid % % decrease in
linoleic acid
Palmitic
acid %
% decrease in
palmitic acid
HOP-IL_MAS-108 75.3 35.1 6.6 72 7.9 0.4
HOP-IL_MAS-109 75.6 35.7 5.7 76 7.6 0.4
HOP-IL_MAS-111 79.3 42.3 3.1 87 7.3 0.5
HOP-IL_MAS-116 79.7 43.0 3.5 85 6.8 0.5
HOP-IL_MAS-119 78.5 40.9 3.9 83 6.6 0.6
HOP-IL_MAS-120 74.8 34.2 7.0 70 7.1 0.5
HOP-IL_MAS-123 61.0 9.4 19.4 17 10.0 0.1
HOP-IL_MAS-125 78.2 40.4 5.8 75 7.1 0.5
HOP-IL_MAS-130 80.5 44.5 2.7 88 6.6 0.6
HOP-IL_MAS-138 79.8 43.3 2.7 89 7.2 0.5
HOP-IL_MAS-144 58.3 4.7 21.5 8 10.0 0.1
HOP-IL_MAS-145 80.3 44.2 2.6 89 7.1 0.5
HOP-IL_MAS-154 75.9 36.2 7.2 69 7.8 0.4
HOP-IL_MAS-163 72.2 29.6 9.1 61 7.9 0.4
HOP-IL_MAS-164 56.7 1.8 22.8 3 10.5 0
HOP-IL_MAS-166 63.8 14.5 16.9 28 9.3 0.2
HOP-IL_MAS-171 69.1 24.0 12.3 48 8.3 0.3
HOP-IL_MAS-172 78.6 41.1 4.1 83 6.7 0.5
HOP-IL_MAS-181 75.0 34.6 7.3 69 7.5 0.4
HOP-IL_MAS-191 79.8 43.2 2.7 89 6.7 0.5
HOP-IL_MAS-201 79.1 41.9 4.5 81 6.6 0.6
ICGV 05141 55.7 0 23.4 0 10.8 0
123
162 Page 10 of 15 Euphytica (2018) 214:162
using marker-assisted breeding. Realizing the impor-
tance of oil content for Indian consumers, the high oil
content feature of the female parent has been success-
fully retained in majority of the recombinant lines.
Furthermore, back ground selection with selected
SSRs revealed the development of ideal recombinants
carrying genomic region of female parent adjacent to
ahFAD2 loci along with high oleic acid as well as high
oil content. Thus, we successfully transferred the high
oleic acid content from SunOleic 95R in the genetic
background of ICGV 05141, which resulted in devel-
opment of recombinants of ICGV 05141 with high
oleic acid. Up to 44% increase in oleic acid content
was observed in the recombinant lines as compared to
ICGV 05141. The increase in oleic acid content in
recombinant lines simultaneously reduced the levels
of linoleic acid. This was due to mutation in both the
ahFAD2 loci resulting in less/no production of fatty
acid desaturase enzyme which stopped conversion of
oleic acid to linoleic acid. Reduced linoleic and
palmitic acid contents have additional health benefits
to the consumers. However, so far available studies or
data do not allow determination of the level of dietary
linoleic acid needed for optimum health (Jandacek
2017). Linoleic acid in recombinant lines decreased up
to 89% as compared to female parent. Similarly,
palmitic acid in recombinant lines decreased up to
0.6% as compared to female parent. Thus, low linoleic
acid and low palmitic acid traits have also been
introgressed from SunOleic 95R. Studies concerning
high oleic acid have mostly focused on the levels of
oleic acid and linoleic acid in the recombinant lines.
Very often it was observed that the change in one
metabolite brought about by a change in the
corresponding enzyme in a biosynthetic pathway,
affected the levels of all other metabolites in the
pathway. Earlier studies by Pandey et al. (2012) and
Wang et al. (2015b) illustrated the effect of ahfad2
alleles on palmitic acid levels. Here, we also observed
significant reduction in palmitic acid simultaneously
with the increase in oleic acid content in recombinant
lines due to introgression of ahfad2 alleles. Moreover,
oil, oleic acid, linoleic acid and palmitic acid contents
traits are quantitative in nature and would vary with
the change in environments (Sarvamangala et al.
2011). The variation observed here in oil content
among recombinant lines could be due to geno-
type 9 environmental interactions. Besides, Knauft
et al. (1993) and Moore and Knauft (1989) reported
that inheritance of high-oleate trait in F-435 mutant is
controlled by two recessive genes. Later, Isleib et al.
(1996) reported that oleic acid content in Virginia
peanut was controlled by two loci but with modifiers
and additional epistatic interactions. This could be one
of the possible reason for variation in oleic acid (56 to
80%) content in recombinant lines.
Yield superiority and yield stability of a genotype
over environments are two key parameters of a success-
ful variety (Allard 1960). The genotypic selection
combined with phenotypic selection was found effective
in selecting recombinant lines with target traits, desired
plant features and agronomic value. The recombinant
lines were initially selected by genotyping and later
tested for phenotypic traits, biochemical parameters, and
yield. Selected 10 recombinant lines yielded either
significantly higher or at par with female parent.
Moreover, shelling percent and hundred-kernel weight
of these recombinant lines were also either higher or at
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
O/L
Fig. 7 Oleic to linoleic acid
ratio in recombinant lines
and parents pooled over
2013 rainy and 2013 post
rainy seasons
123
Euphytica (2018) 214:162 Page 11 of 15 162
Table
3Yield
andrelatedtraits
ofrecombinantlines,ICGV
05141andGG
20
Recombinantline
2014rainyseason
2015rainyseasons
Pooled
Pod
yield/plota(g)
Shelling(%
)100-kernel
weight(g)
Pod
yield/plota(g)
Shelling(%
)100-kernel
weight(g)
Pod
yield/plota(g)
Shelling(%
)100-kernel
weight(g)
HOP-IL_MAS-108
948
70
27
800
72
27
874
71
27
HOP-IL_MAS-109
750
69
26
688
73
24
719
71
25
HOP-IL_MAS-111
1032
75
26
1200
73
28
1116
74
27
HOP-IL_MAS-116
1400
70
30
1750
74
26
1575
72
28
HOP-IL_MAS-119
644
68
27
800
66
27
722
67
27
HOP-IL_MAS-120
1352
66
28
1100
68
28
1226
67
28
HOP-IL_MAS-123
980
75
26
1248
73
30
1114
74
28
HOP-IL_MAS-125
1218
72
32
1400
72
32
1309
72
32
HOP-IL_MAS-130
1782
70
30
1500
72
36
1641
71
33
HOP-IL_MAS-138
1132
70
35
1200
70
31
1166
70
33
HOP-IL_MAS-144
1500
69
39
1344
73
39
1422
71
39
HOP-IL_MAS-145
2064
68
29
2100
66
27
2082
67
28
HOP-IL_MAS-154
1028
70
31
900
70
35
964
70
33
HOP-IL_MAS-163
2100
72
28
2002
68
28
2051
70
28
HOP-IL_MAS-164
1094
65
28
1400
65
22
1247
65
25
HOP-IL_MAS-166
900
75
32
762
71
32
831
73
32
HOP-IL_MAS-171
1426
74
35
1600
74
33
1513
74
34
HOP-IL_MAS-172
1500
71
35
1378
71
41
1439
71
38
HOP-IL_MAS-181
2004
66
26
2200
64
26
2102
65
26
HOP-IL_MAS-191
2002
74
25
2300
68
31
2151
71
28
HOP-IL_MAS-201
1400
68
25
1700
66
25
1550
67
25
ICGV
05141
1606
68
35
1300
68
41
1453
68
38
GG
20
1200
68
43
1446
64
35
1323
66
39
CD
at5%
239.77
2.44
2.61
294.25
2.34
2.53
125.07
2.45
2.45
CV%
10.75
2.23
4.63
12.81
2.16
4.12
5.67
2.12
4.94
aPlotsize
=7.20m
2
123
162 Page 12 of 15 Euphytica (2018) 214:162
par with the female parent. Besides, passport data
reiterates that qualitative and quantitative traits of
recombinant lines and ICGV 05141 did not vary much
except oleic acid content. Thus, recombinant lines, bred
here using marker assisted selection by cutting huge
resources and time, would be high yielding peanut
cultivar(s) with high oil and high oleic acid content to the
peanut farmers of India.
Conclusion
Peanut with desirable fatty acid composition is key for
sustaining the demand of consumers and traders in
coming years realizing the tough competition from
other oil crops. India being the second largest peanut
producer holds great market potential of high oil and
oleic peanut and therefore, we developed recombi-
nants of ICGV 05141 with high oleic acid besides high
oil, pod yield and yield related traits devoid of much
time and resources. These recombinant lines will
certainly extend additional economic benefits to the
Indian farmers in addition to ensuring availability of
high oleic peanuts to the traders and industry vis-a-vis
peanut oil and other food products with extended shelf
life and additional health benefits to the consumers.
Acknowledgements Authors acknowledge the financial
support received vide no. 11-2/2010-Pul (TMOP), Govt. of
India, Ministry of Agriculture, Department of Agriculture and
Cooperation.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
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